Epigenome-Microbiome crosstalk: A potential new paradigm influencing neonatal susceptibility to disease

<p>Preterm birth is the leading cause of infant morbidity and mortality. Necrotizing enterocolitis (NEC) is an inflammatory bowel disease affecting primarily premature infants, which can be lethal. Microbial intestinal colonization may alter epigenetic signatures of the immature gut establishing inflammatory and barrier properties predisposing to the development of NEC. We hypothesize that a crosstalk exists between the epigenome of the host and the initial intestinal colonizing microbiota at critical neonatal stages. By exposing immature enterocytes to probiotic and pathogenic bacteria, we showed over 200 regions of differential DNA modification, which were specific for each exposure. Reciprocally, using a mouse model of prenatal exposure to dexamethasone we demonstrated that antenatal treatment with glucocorticoids alters the epigenome of the host. We investigated the effects on the expression profiles of genes associated with inflammatory responses and intestinal barrier by qPCR-based gene expression array and verified the DNA modification changes in 5 candidate genes by quantitative methylation specific PCR (qMSP). Importantly, by 16S RNA sequencing-based phylogenetic analysis of intestinal bacteria in mice at 2 weeks of life, we showed that epigenome changes conditioned early microbiota colonization leading to differential bacterial colonization at different taxonomic levels. Our findings support a novel conceptual framework in which epigenetic changes induced by intrauterine influences affect early microbial colonization and intestinal development, which may alter disease susceptibility.</p

MetaCore gene enrichment analysis of ileum microarray data representing microbiota effects in preweaned M_PI-L and M_PI-H-gnotobiotic mice.

<p>M_<b>PI</b>-L and M_<b>PI</b>-H induced differentially expressed genes were analyzed by GeneGO MetaCore software. Panel A: Top 10 overrepresented GO biological processes in preweaned M_PI-L, H vs GF mice. Panel B: Top 10 scored overrepresented networks in preweaned M_PI-L, H vs GF mice. Panel C: Comparison of the differentially expressed genes in M_PIL_GF and M_PI-H_GF presented in inflammatory response-related networks using the IPA program. Orange line: activation; Blue line: inhibition; Grey: not present.</p

NF-κB activation in M_PI-L, -H and SPF GF ileum mucosa by immunohistochemistry.

<p>A: NF-κB activation was analyzed by immunohistochemical staining of 5 micron paraffin-embedded distal ileum tissue from GF, M_PI-L, H and SPF mouse. Representative areas (n = 3/per group) are shown (magnification 400x), black arrows indicate nuclear translocation of the phosphorylated NF-κB p65 subunit. B: Nuclear translocation of pp65 was quantified by ImmunoRatio tool from Image J software and presented as % of pp65 positive nuclei/total nuclei. C: Growth curve of M_PI-L (n = 20) and M_PI-H (n = 15) from birth to weaning. One-way ANOVA with post-hoc Tukey’s HSD test was used to compare the groups.</p

M_PI effects on systemic immune and inflammatory cytokine expressions by multiplex immunoassay.

<p>Multiplex immunoassay of immune and inflammatory cytokine expressions in serum collected from preweaned GF and M_PI-L, H and SPF. The levels of these cytokines are presented as mean ± SEM. One-way ANOVA with post-hoc Tukey’s honest significance (HSD) test was used to compare the groups.</p

Comparison of bacterial communities following transfaunation of germ free mice with M_PI fecal lysates.

<p>A: The relative abundance of different bacterial phyla in input M_<b>PI</b>-L,-H fecal samples and from M_PI-L, H and SPF preweaned mouse pups is shown. B: PCoA analyses of the M_PI-L and M_PI-H in original human infants, transfaunated dams and respective pups based on Bray Curtis dissimilarities among samples given presence/absence of major taxa present in at least one sample. Axis 1 explained 27.5% of variation and axis 2, 18.1%.</p

The composition of microbiota in M_PI-L and M_PI-H.

<p>The unique bacterial species are in Italic.</p><p>The composition of microbiota in M_PI-L and M_PI-H.</p

AvrA expression stabilizes the protein expressions of occludin and ZO-1 <i>in vitro</i>.

<p>(A) Western blot of occludin-1, ZO-1, claudin-1, and E-cadherin. Polarized human colonic epithelial T84 cells were colonized with AvrA-deficient or -sufficient bacterial strains for 30 minutes, washed with HBSS and incubated in DMEM for 30 minutes. Cells were lysed. Equal volumes of total cell lysate were processed for immunoblotting with Rabbit anti-claudin-1, Mouse anti-occludin-1, Mouse anti-ZO-1 antibodies, or E-cadherin antibodies. Experimental groups: Control: polarized T84 cells without any treatment; WT: wild-type <i>S</i>. Typhimurium ATCC 14028s without sufficient AvrA protein expression; PhoP^c: parental PhoP^c with sufficient AvrA protein expression; AvrA⁻: PhoP^c AvrA mutant; AvrA⁻/AvrA⁺: PhoP^c AvrA- transcomplemented with a plasmid encoding WT AvrA; or <i>E.coli</i> F18: commensal bacteria isolated from human intestine. (B) Densitometry of occludin and ZO-1. Relative occludin-1 and ZO-1 band intensity was determined using NIH Image 1.63 software. Occludin-1 and ZO-1 expression significantly increased in the PhoP^c group compared to the Control, WT, and AvrA- groups in absence of AvrA protein. * P<0.05. Data are reported as mean±SD of 3 independent experiments.</p

Immunostaining of claudin-1 and ZO-1 <i>in vivo</i>.

<p>Immunostaining on mouse colonic epithelial cells was performed 24 hours after mouse infection with PhoP^c, AvrA⁻ or AvrA⁻/AvrA⁺. Experimental groups: Control: normal mouse cells; PhoP^c: mice infected with parental PhoP^c with sufficient AvrA protein expression; AvrA⁻: mice infected with PhoP^c AvrA mutant. Tissues were fixed, permeabilized, and stained with claudin-1 and ZO-1 antibodies, followed by A488 secondary antibodies, A594 secondary antibodies, and DAPI. AvrA⁻ infected mice display disruption of the TJ structure. Arrows in Panel ZO-1 show the red staining of ZO-1 protein on the top of the intestinal crypts. White arrow in Panel AvrA- DAPI shows lymphoid aggregation. Please note disorganized structure of ZO-1 in the colonic epithelial cells infected with AvrA- bacterial strain. Images shown are from a single experiment and are representative of three separate experiments. <i>n</i> = 3 animals in each experimental group.</p

Immunostaining of claudin-1 and ZO-1 in cells colonized with AvrA-sufficient or -deficient bacteria <i>in vitro.</i>

<p>T84 monolayers were treated with PhoPc or AvrA-. After 8 hours, the monolayers were fixed and immunostained for claudin-1 and ZO-1. ZO-1 distribution in the control cells without any treatment has its normally smooth nature. In PhoP^c-treated cells, the distribution of ZO-1 was very similar to that in the control cells. ZO-1's appearance in PhoP^c group was similar as the control group when cells were viewed in cross-Z-section (Z-section for Control and PhoP^c). However, in cells treated with <i>Salmonella</i> derivative AvrA- mutant (without AvrA), the normally smooth arc-like ZO-1 profiles were transformed into a complex series of irregular undulations (first row of panels AvrA-). ZO-1 staining became thinner and more sinuous. The Z-section panel in Fig. 3 shows the weak staining of ZO-1 in AvrA-. AvrA absence induced a disorganization of transmembrane protein claudin-1, and the protein was moreover expanded intracellularly (second row, see arrow). PhoP^C treatment also slightly changed the distribution of claudin-1. Intracellular claudin-1 was detectable in the cytosol of the cells colonized with PhoP^c. Results are representative of 5 independent experiments.</p

The <i>Salmonella</i> AvrA protein modulated ZO-1, occludin, and claudin-1 expression <i>in vivo</i>.

<p>Mice were infected with <i>bacteria</i> for 18 hrs and intestinal epithelial cells were harvested for ZO-1, claudin-1, occludin, and α-catenin expression by immunoblot. Experimental groups: C: normal mouse cells; WT: wild-type <i>S</i>. Typhimurium ATCC 14028s without sufficient AvrA protein expression; PhoP^c: parental PhoP^c with sufficient AvrA protein expression; AvrA⁻: PhoP^c AvrA mutant; PhoP^c AvrA⁻/AvrA⁺: PhoP^c AvrA- transcomplemented with a plasmid encoding WT AvrA; or <i>E.coli</i> F18: commensal bacteria isolated from human intestine. Images shown are from a single experiment and are representative of three separate experiments.</p